The invention is in the general field of analyte detection, assays, and methods for the separation of particular compounds from a mixture.
Assays of Biomolecules: Assays can be used to determine whether, and how much of, an analyte is present in a sample. In some cases, such assays rely on selective binding or complexation (specific or nonspecific) of the analyte in the sample with an exogenously supplied capture reagent or binding partner.
Effects of endogenous binding partners on assays: Often such samples contain an endogenous component that forms a complex with the analyte, and the resulting endogenous complex may interfere with detection of the analyte. For example, detection by absorbance, fluorescence, molecular weight, or other analyte characteristics may be adversely affected by endogenous complexes. Where detection itself depends on formation of a complex with an exogenously supplied reagent, analyte present in endogenous complexes may be unable to effectively complex with the exogenous binding partner (as is required for detection in such assays). In that way, the endogenous complex interferes with the assay""s reliability. For example, the analyte goes undetected or is incompletely detectedxe2x80x94i.e., it provides a false negative or non-quantitative result.
This problem can be illustrated with standard enzyme-linked immunosorbent assays (ELISAs), in which sample antigen is detected only if it is recognized and bindable by immobilized antibody. Endogenous sample antibodies that react with the analyte may prevent at least some portion of the analyte from complexing with one or both of the exogenous assay reagents (antibodies), thereby reducing the effectiveness of the assay.
Assays for antigens or antibodies that are characteristic of a pathogen are particularly susceptible to problems caused by endogenous binding partners. If the patient being assayed has developed an immune response to the analyte antigen, a significant portion of sample antigen may be present in undetectable endogenous antigen/antibody complexes. Similarly, in serology assays where the antibody is the analyte to be measured, some of the sample antibody that the assay is designed to measure may be complexed with endogenous pathogen antigen.
In addition to endogenous antibody/antigen complexes, other endogenous complexes can interfere with assays, for example, various serum globulins can interfere with immunoassays for thyroxine, estradiol, cortisol, and testosterone. See, Thorell, J. I., and Larson, S. M, in xe2x80x9cRadioimmunoassay and Related Techniques,xe2x80x9d C. V. Mosby, St. Louis, 1978. Vitamin B12 assays are perturbed by the binding of transcobalamin. See, Laue et al. Blood 26:202 (1965). Immunoassays for prostate-specific antigen (PSA) are perturbed by endogenous complexes with a serine protease inhibitor, xcex11-antichymotrypsin. See, Lilja et al. Clin. Chem. 37:1618-1625 (1991).
Another area in which endogenous complexes may seriously affect assay results is the use of tumor antigens to mark the tumor""s presence, e.g. in an immunoassay. Frequently, these tumor markers may be masked by endogenous complexes. For example, serum thyroglobulin autoantibody interferes with detection of differentiated thyroid carcinoma. Another example of the difficulty of obtaining accurate quantitation of a serum tumor antigen is the epithelial mucin MUC-1. Gorevitch et al., Br. J. Cancer, 72:934-938 (1995); and Hilgers et al. Scand. J. Clin. Ob. Invest. Suppl., 221:81-86 (1995).
A particular problem which may be related to endogenous complex formation has surfaced in HIV assays. Tsiquaye et al., AIDS, 2:41-45 (1988); McHugh et al., J. Infect. Dis., 158:1088-1091 (1988); Nishanian et al., J. Infect. Dis., 162:21-28 (1988); and Carini et al., Scand. J. Immuno., 26:1 (1987). Other assays in which this problem can arise include: epithelial mucin (MUC-1 and PEM) assays (Gorevitch et al., Br. J. Cancer, 72:934-938 (1995); and Hilgers et al. Scand. J. Clin. Ob. Invest. Suppl., 221:81-86, 1995); Hi histones in assays for systemic lupus (Wesierska-Gadek et al. Arthritis Rheum, 33:1273-1278, 1990); assays for the tuberculosis pathogen (Dlugovitzky et al. Braz. J. Med. Biol. Res. 28:331-335, 1995); alpha-fetoprotein assays to detect hepatocellular carcinoma (Tsai et al., Br. J. Cancer, 72:442-446, 1995); assays for Yersinia enterocolitca and Yersinia pseudotuberculosis (Didenko et al. J. Basic Microbiol. 35:163-170, 1995); and assays for the leprosy pathogen (Sinha et al. Int. J. Lepr. Other Mycobact. Dis., 60:396-403, 1992).
Various methods have been described to dissociate endogenous antibody/antigen complexes and thereby to improve assay sensitivity, including solvent extraction, heating, protein precipitation, use of competitive inhibitors, and pH changes. For example, Mosier, U.S. Pat. No. 4,656,251, and Weil et al., J. Immunology, 134:1185-1191 (1985), disclose pretreating a canine sample to break up immune complexes before assaying for heartworm antigens. Mosier ""251 discloses a process that includes acidification to dissociate the complex, followed by heating to denature dissociated antibodies. Weil discloses (p. 1186, right column) a process including the addition of EDTA followed by heating.
Accelerating High Sensitivity Assays: While sensitivity may be improved by lengthening incubation time (e.g., overnight), high throughput and automation are also important goals that may be inconsistent with lengthy incubation. As high throughput automated instruments have become widely utilized, assay results are needed more quickly (i.e., within a few minutes). The need to accelerate analyte/binding partner interactions may be addressed by adding a large excess of the exogenous binding partner, or by using temperature conditions above optimum to drive the binding reaction as far as possible in an acceptable assay time.
Separation of Biomolecules: A widely accepted method for purification of bioactive compounds is affinity chromatography. This method is based on the premise that many bioactive compounds bind to other molecules with extraordinary specificity. These other molecules are commonly referred to as xe2x80x9cligands.xe2x80x9d For example, ligands that-have been identified for binding specific compounds include, but are not limited to, nucleic acids, vitamins, carbohydrates, fats, and proteins (e.g., enzymes, antibodies, and receptors).
The first step in affinity chromatography typically is identification of a ligand that binds specifically to the compound of interest. Such ligands are already known for many enzymes and other compounds. Once a ligand has been identified and obtained, the ligand can be attached to a solid support. The solid support can, for example, be trapped within a porous sack or, more commonly, immobilized in a porous column. A solution known to contain, inter alia, the compound of interest is generally flushed through the column so that the solution comes into binding contact with the immobilized ligand.
The quantity of immobilized ligand required depends on the amount of the desired compound expected to be present. Typically, each ligand can bind to a limited number of (e.g., often one) molecules of the compound. Numerous complications render this generalization less valid in practice, however. For example, steric constraints can limit the number of molecules of the compound that can exist within a given volume, especially if the compound is, for example, a relatively large molecular weight, multi-domain protein. Also, there can be other, undesirable compounds capable of weakly binding to the same ligand that the compound of interest binds tightly to. The latter problem can become especially acute if the undesired weakly binding compound is present in excess (i.e., relative to the desired compound). Therefore, it is desirable to promote high affinity, high specificity interactions.
In a typical preparative application of affinity chromatography, an impure solution containing the desired compound is passed through a porous material (e.g., in a bed or column) containing the immobilized ligand. The desired compound becomes bound to the ligand and therefore is itself immobilized. The remaining impurities that were in the solution, including other compounds, are washed away with an additional fresh buffer or solvent, leaving the immobilized ligand bound to the desired compound.
Once the impurities have been washed away, the compound of interest can be released from the binding relationship with the immobilized ligand. This process is called xe2x80x9celution.xe2x80x9d Elution can be effected by making the compound-ligand complex unstable, for example, with altered pH, temperature, or ion concentration, or by adding a different ligand known to have still greater affinity for the compound relative to the immobilized ligand (i.e., to displace the compound). It is desirable to effect elution with relatively mild conditions to avoid irreversible damage to either the ligand or the desired compound. It is also desirable to elute the desired compounds under conditions that ensure simple recovery following separation.
Affinity chromatography sometimes uses antibodies, enzymes, or other binding proteins as the immobilized member of the binding pair, with the specific ligand or substrate being the desired compound to be isolated or purified. The type of affinity chromatography termed immunoaffinity chromatography uses antibodies as ligands. Some advantages of immunoaffinity chromatography are that: 1) the immunological process of antibody diversity does the work of finding a ligand, and 2) antibodies exhibit high specificity.
The invention features: (1) pressure-mediated dissociation of an analyte complexed with an endogenous binding partner to enable detection of a complex formed from the analyte and an exogenous binding factor, (2) pressure-mediated association of an analyte and an exogenous binding partner to enable more rapid and/or more sensitive detection of an analyte, and (3) pressure-mediated association and dissociation of biomolecular complexes to enable separation of one biomolecule from a complex mixture.
Pressure can be used to improve assays by dissociating endogenous analyte complexes and improving assay speed and sensitivity by associating the analyte molecules with exogenously supplied binding partners. Pressure can also be used to improve the separation of compounds from contaminated mixtures.
Assays: Pressure can be controlled to dissociate endogenous analyte complexes and thereby improve detection of analyte present in samples containing endogenous components that complex with the analyte. As described above, such endogenous complexes can interfere with analyte detection in various ways. One specific example of such interference involves detection formats that rely on a determination of complexing between analyte and an exogenously supplied specific binding partner for the analyte.
In one aspect of the invention, endogenous analyte complexes are dissociated under controlled pressure to improve analyte availability for detection. For example, pressure-induced dissociation of the endogenous complexes (weak or strong) can improve analyte detection by improving binding (i.e., kinetically or thermodynamically) to the exogenous binding partner. This aspect of the invention features a dissociation step in which the sample is subjected to elevated pressure sufficient to dissociate an endogenous complex formed from an analyte and an endogenous sample component (e.g., preferably at least 15,000 psi, i.e., 105 MPa, and most preferably at least 30,000 psi, or 210 MPa). This dissociation step is followed by an analyte detection step, e.g., in which the exogenous specific binding partner is reacted with sample analyte. In this format of the invention, it is believed that increasing the pressure results in structure disruption (either a reversible or irreversible change in three-dimensional conformation) of a component of the endogenous complex which prompts binding partner dissociation.
More specifically, the dissociation pressure can (but need not necessarily) be high enough to irreversibly dissociate the analyte from endogenous binding component. If dissociation is irreversible (for example, because one member of the complex is structurally disrupted in a way that substantially prevents endogenous complex reassociation), then the assay step can be performed without first removing the structurally disrupted binding component from the analyte. If desired, an agent that prevents or reduces reassociation can be added, such as a denaturing agent (e.g., urea); a water miscible solvent; a chelating agent, such as EDTA, EGTA, or o-phenanthroline; a detergent; or a chaotrope, such as dithiothreitol, urea, or thiocyanate. This agent or agents preferably should be tolerated in subsequent assay steps, so that it need not be removed prior to those steps.
For reversible dissociation, the analyte is removed from the endogenous sample component in a separate step performed after the dissociation step. For example, the exogenous analyte binding reagent can be immobilized in a chamber with the sample; chamber pressure is increased to dissociate the complex, after which the endogenous binding component is removed from the chamber while pressure is maintained. The chamber can be a semipermeable membrane selected to pass endogenous analyte, but not endogenous sample component.
In another embodiment (or in a combination of the above described embodiments), temperature, pressure, or both, are controlled (usually increased) to control the association between a ligand present in a mixture and an exogenously supplied binding partner. For example, an assay is performed where pressure and temperature are maintained to improve association compared to ambient (1 atm, room temperature) conditions. It is possible to combine the various aspects of the invention by first subjecting a mixture to a temperature and pressure for separating a ligand (analyte) in the mixture from endogenous binding partners in the mixture, and then changing temperature or pressure, or both, of the separated ligand to a second temperature or pressure, or both, selected to enhance ligand complex formation relative to complex formation at ambient temperature and pressure. Generally, the second temperature or pressure is intermediate between ambient conditions and the temperature and pressure used in the separating step above. While at the second temperature and pressure, the separated ligand is reacted with the exogenously supplied binding partner.
The method can be performed using apparatus (described in PCT/US96/03232, incorporated by reference) in which a valved inlet connects the chamber to a pressurized supply area, a valved outlet connects the chamber to a waste collection area, and controllers operate the valved outlets. Analyte is flushed out of the chamber and into the collecting chamber by introducing material from the pressurized supply area.
The invention can be practiced with a wide diversity of analytes. In particular, the analyte may be an antigen, and the method may be used to dissociate an endogenous antibody that complexes with the antigen. Where the analyte is an antibody, the method can be used to dissociate a complex between an antigen and the antibody. In addition, where the analyte is complexed with multiple endogenous components, the method can be used to dissociate the analyte from such one or more of these complexes.
Such dissociation is particularly useful where endogenous complexes are known to interfere at least to some extent with assaying. The invention can specifically be used to assay: a) HIV antigens (e.g., p24, gp41, gp120, gp160, and p15) where the sample is a human bodily fluid containing anti-analyte antibody; b) non-protein analytes, such as thyroxine, estradiol, cortisol, and testosterone, where the sample is a bodily fluid comprising serum globulin; c) Vitamin B12 where the sample contains transcobalamin; d) prostate-specific antigen where the sample contains xcex11-antichymotrypsin or xcex12-macroglobulin; e) an epithelial mucin, where the sample contains endogenous antibody that complexes with the analyte; f) antibody to Hi histones in an assay for systemic lupus, where the sample contains endogenous H1 histones; g) tuberculosis pathogen, where the sample contains anti-analyte antibodies; h) alpha-fetoprotein where the method is a diagnostic for hepatocellular carcinoma; i) an antigen of Yersinia enterocolitca or Yersinia pseudotuberculosis; j) an antigen of the leprosy pathogen, Mycobacterium leprae; k) anti-DNA antibodies or DNA binding thereto; 1) Dirofilaria immitis antigen or antibody thereto; m) growth hormone or growth hormone-binding protein; n) cholesterol; o) low density lipoprotein; p) high density lipoprotein; and q) tumor antigens that can be used as diagnostic, monitoring, or prognostic indicators for cancer-related pathological states.
A specific concern with many tumor markers is that although they are often detected in benign disease, they may be absent in early-stage malignancy, due to complex formation with a patient""s antibodies. For example, serum thyroglobulin autoantibody interferes with detection of differentiated thyroid carcinoma. With proper controls and dissociation of complexes, tumor markers may be used to quantitate tumor burden, e.g., to monitor clinical progress and patient status. In particular, tumor antigen burden can be measured serially over time. The invention also permits separation of total analyte levels into dissociated analyte and analyte present in endogenous complexes. The information thus derived, e.g., autoantibody levels, can be useful clinically. Where free antigen and complexed antigen appear out of balance, the balance can be corrected extracorporeally to increase both cellular and humoral cytotoxicity to the tumor.
Not only does the invention improve assays involving endogenous complexes, it also makes possible a better understanding of precisely how much and what type of antigens and antibodies are present, regardless of whether they are present in complexes. This capability can improve the ability to track the immune response to, and the course of, a disease.
The invention may also improve assays in which the endogenous analyte is not initially complexed with the analyte, but the assay protocol and reagents induce formation of such an undesired complex. The term xe2x80x9cendogenous complexxe2x80x9d includes such undesired complexes which are induced by the assay, even complexes which form with assay components; so long as the complexes are not the desired complex which results in a detectable event. Pressure control can also ameliorate such de novo assay-induced complex formation.
The invention also includes an embodiment wherein the dissociation of endogenous binding complexes is accompanied by association of an exogenous binding partner such as an aptamer.
Separation: Pressure""s influence on affinity can be used to improve affinity chromatography methods. Contaminated mixtures containing compounds of interest can be placed in fluid contact with molecules known to bind to the desired compounds with pressure-dependent affinity or activity. The pressure in the reaction vessel can be altered to allow the desired compounds to bind more quickly or more tightly to the immobilized molecules. The contaminants can be flushed away first, and then the purified compounds can be dissociated from the immobilized molecules, for example by further modifying the pressure.
In general, the invention features a method for separating a desired compound from at least some contaminants in a mixture containing the desired compound and one or more contaminants. The method includes providing binding molecules having pressure-dependent affinity for the desired compound, providing the mixture (i.e., in fluid contact with the binding molecules), and subjecting the molecules and the mixture to a first pressure that increases the affinity of the binding molecules for the desired compound, to form a bound complex. These steps can be performed in any order. The bound complex is then separated from at least some of the contaminants, the pressure is changed to a second pressure (i.e., which decreases the affinity of the binding molecules for the desired compound), and the binding molecules are finally separated from the desired compound.
In the present context, the term xe2x80x9caffinityxe2x80x9d is used to describe both the kinetics and thermodynamics of binding. Thus, when it is said that a set of conditions xe2x80x9cenhances the affinity of an analyte for a reagent,xe2x80x9d it is to be understood either that the conditions enhance the rate of formation of an analyte-reagent complex or that the conditions drive the equilibrium of the reaction system toward complex formation, or both. xe2x80x9cEnhanced ligand complex formationxe2x80x9d would have the same meaning.
Furthermore, the phrase xe2x80x9cmolecules that associate with a compound in a pressure-dependent mannerxe2x80x9d means that either the rate of the association (i.e., binding molecule-desired compound complex formation) is increased or the equilibrium of the system is shifted toward association, or both.
The first and second pressures can each be uniform throughout the system that includes both the binding molecules and the mixture in fluid contact with the molecules.
The binding molecules can be immobilized, for example by attachment to a solid support (e.g., a polymer bead, a particle, a strip, a tube, a column, a molded material, and a polymer matrix) or by using a semipermeable membrane that passes only one of the components of the binding pair.
The fluid mixture can include, for example, water, an aqueous solution, an organic solvent, an organic solution, a gas, or a supercritical fluid.
The desired compound and the binding molecules can include, but are not limited to, members of the following classes which can form bound complexes: polypeptides, proteins, antigens, haptens, antibodies, prions, carbohydrates, nucleic acids, steroids, triglycerides, substrates, enzymes, and hormones. Thus, the bound complex can be, for example, an enzyme-substrate complex, a ligand-receptor complex, a glycoprotein-lectin complex, a protein-cofactor complex, a nucleic acid-cofactor complex, a hybridized nucleic acid-target complex, a hapten-antibody complex, or an antigen-antibody complex.
The first pressure, or both the first and second pressures, can be greater than atmospheric pressure (e.g., between 500 and 200,000 psi or between 5,000 and 200,000 psi).
The first pressure can be applied prior to or after providing the mixture, the binding molecules, or both.
pH, ionic concentration, fluid composition, or temperature can be modified to enhance separation of the compounds of interest from the binding molecules. Additionally, a reagent can be added to cause dissociation of the bound complex to yield the free compound of interest and the unbound molecules. The reagent can be, for example an acid, a base, a salt, a metal, a metal-scavenger, a detergent, a dissociating agent, a chaotropic agent, water, an organic solvent, a chelating agent, or some other binding partner. More specifically, such a reagent can be, for example, a magnesium salt, a lithium salt, sodium dodecyl sulfate, urea, guanidine hydrochloride, thiocyanate, or dioxane.
The desired compound can be, for example, an enzyme (wherein the binding molecules can be substrates for the enzyme); an antibody, such as a monoclonal antibody (wherein the binding molecules can be antigens for the antibody); or a cofactor, such as a transcription cofactor (wherein DNA transcription can be modulated).
The three-dimensional conformation of the desired compound can, for example, change in the transition from the first to the second pressure.
In another embodiment, the invention features another method for separating a compound of interest from at least some contaminants in a mixture containing the desired compound and one or more contaminants.
This method includes providing binding molecules having affinity for the desired compound and further having pressure-dependent activity for the modification of the desired compound. This means that the binding molecules not only bind to the desired compound, but also cause a modification of the compound. An example of such a system is provided by an enzyme and its substrate. At atmospheric pressure, many enzymes bind to their substrates, modify the substrates to generate xe2x80x9cproducts,xe2x80x9d then release the products.
The modifying activity of many such enzymes is attenuated by elevated pressure, while binding affinity is often enhanced by pressure. Thus, the enzyme""s ability to bind to the substrate can be exploited for purification, provided that the pressure is not lowered to a level at which the enzyme exhibits modifying activity.
The method of this embodiment also includes providing the aforementioned mixture in fluid contact with the binding molecules to form a bound complex at a first pressure that decreases the activity of the binding molecules for the modification of the compound. These two steps can be executed in any order. The bound complex is then separated from at least some of the contaminants.
In some examples, the pressure is changed to a second pressure (i.e., that increases the activity of the binding molecules for the modification of the compound) and the unbound molecules are finally separated from the modified compound (i.e., the product).
Alternatively, or additionally, pH, ionic strength, fluid composition, or temperature can be modified to enhance separation of the compounds of interest from the unbound molecule.
In another alternative method, a reagent that causes dissociation of the bound complex is provided, and the binding molecules are separated from the desired compound. Examples of such reagents include acids, bases, salts, metals, metal-scavengers, detergents, dissociating agents, chaotropic agents, water, organic solvents, chelating agents, and other binding partners. Specifically, the reagent can be, for example, a magnesium salt, a lithium salt, sodium dodecyl sulfate, urea, guanidine hydrochloride, thiocyanate, or dioxane.
The first pressure can be uniform throughout the system that includes the binding molecules and the mixture in fluid contact with each other.
The binding molecules can be immobilized, for example, by compartmentalization within a semipermeable membrane or by attachment of the binding molecules to a solid support (e.g., a polymer bead, a particle, a strip, a tube, a column, a molded material, or a polymer matrix).
The fluid mixture can be, for example, water, an aqueous solution, an organic solvent, an organic solution, a gas, or a supercritical fluid.
The desired compound can be, for example, a polypeptide, a nucleic acid molecule, an antibody, a triglyceride, a steroid, a prion, or a carbohydrate.
The binding molecules can be, for example, enzymes, wherein the bound complex can be an enzyme-substrate complex.
Alternatively, the desired compound can be, for example, an enzyme, wherein the binding molecules can be substrates for the enzyme.
The first pressure can be greater than atmospheric pressure (e.g., between 500 and 200,000 psi or between 5,000 psi and 200,000 psi). The first pressure can be applied prior to providing the mixture. The second pressure can also be greater than atmospheric pressure.
In any embodiment, the method can be repeated at least once (e.g., 1, 2, 3, or more times) to remove more of the contaminants from the mixture.
Screening: In another embodiment, the invention features a method of screening a molecular library for molecules that bind a target. The method includes the steps of providing a member of the molecular library in fluid contact with-the target at atmospheric pressure to form a complex; using a detection means to monitor the binding rate and affinity in real-time; subjecting the complex to an elevated pressure that causes dissociation of the complex; flushing the dissociated member away from the immobilized target; repeating these steps for other members of the library; and then analyzing the results collected by the detection means to determine which members of the library bind to the target.
The library can, for example, include proteins, carbohydrates, antibodies, ribozymes, oligonucleotides, peptides, or small organic molecules. The target can be, for instance, a phage display or other immobilized biomolecules. The detector means can be, for example, a radioisotopic detector, an infrared spectrometer, a mass spectrometer, a gas chromatograph, a spectrophotometer, a spectrafluorometer, an electrochemical detector, a surface plasmon resonance detector, a nuclear magnetic resonance spectrometer, a scanning tunneling microscope, an atomic force microscope, or a chemiluminescence spectrometer.
Refolding of Denatured Proteins: In still another embodiment, the invention features a method of refolding a previously denatured protein. The method includes the steps of subjecting aggregates of the denatured protein to elevated pressures sufficient to break up the aggregates to form dissolved, denatured polypeptide chains and then rapidly cycling the pressure to cause the dissolved, denatured polypeptide chains to rapidly sample numerous conformations until the polypeptide chain has folded into its lowest energy protein conformation.
In certain cases, the aggregates are mixed with a pressure-sensitive buffer and contacted with a solid phase having an ionization volume of opposite charge to that of the buffer. The pressure cycling is thought to disrupt the aggregates by pH fluctuation and disperse the aggregates by reversible binding to the solid phase. The buffer itself can be covalently bonded to a solid support (which may or may not be the same as the solid phase), and can also serve as a nucleation site for the refolding of the polypeptide chain. In some cases, reducing and oxidizing agents can be added to allow reconfiguration of disulfide bonds within the polypeptide chain.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
An advantage of the claimed invention is clean separation and isolation of the purified compounds. In certain embodiments, the desired compounds can be eluted from the immobilized binding molecules without addition of supplementary reagents. No packing matrix is necessary either, which means that less of the desired compound is lost through non-specific adsorption as is common in the elution or fractionation steps of traditional chromatographic methods. A packing matrix is a material which can be packed inside a column either to provide a solid phase for the immobilized xe2x80x9ccapturexe2x80x9d reagent or to effect separation by another route such as size exclusion chromatography. The moderate pressures used successfully in the present methods are less likely than the traditional methods to cause irreversible damage either to the desired compounds or to the immobilized binding molecules. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.